Lipopeptides for use in treating liver diseases and cardiovascular diseases

ABSTRACT

The present invention relates to lipopeptide-based compounds for use in the diagnosis, prevention and/or treatment of a liver disease or condition, preferably liver involved metabolic diseases, as well as in the control or modification of the cholesterol level or cholesterol uptake and, thus, diagnosis, prevention and/or treatment of a cardiovascular disease. The present invention furthermore relates to an in vitro or in vivo assay or method for testing or measuring the NTCP-mediated transport of test compound(s). The present invention furthermore relates to a method for the diagnosis, prevention and/or treatment of a liver disease or condition, comprising administering a therapeutically effective amount of a lipopeptide-based compound to a patient. The present invention furthermore relates to a method for the diagnosis, prevention and/or treatment of a cardiovascular disease.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a continuation Application of U.S. patentapplication Ser. No. 14/442,304, filed May 12, 2015; which is theNational Stage Application of International Application NumberPCT/EP2013/073600, filed Nov. 12, 2013; which claims the benefit of U.S.Provisional Patent Applications Ser. No. 61/725,144, filed Nov. 12, 2012and Ser. No. 61/859,476, filed Jul. 29, 2013; all of which areincorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled“SEQ-LIST15Apr15.TXT”, which was created on Apr. 15, 2015, and is 8 KB.The Sequence Listing is incorporated herein by reference in itsentirety.

The present invention relates to lipopeptide-based compounds for use inthe diagnosis, prevention and/or treatment of a liver disease orcondition, preferably liver involved metabolic diseases, as well as inthe control or modification of the cholesterol level or cholesteroluptake and, thus, diagnosis, prevention and/or treatment of acardiovascular disease. The present invention furthermore relates to anin vitro or in vivo assay or method for testing or measuring theNTCP-mediated transport of test compound(s). The present inventionfurthermore relates to a method for the diagnosis, prevention and/ortreatment of a liver disease or condition, comprising administering atherapeutically effective amount of a lipopeptide-based compound to apatient. The present invention furthermore relates to a method for thediagnosis, prevention and/or treatment of a cardiovascular disease.

BACKGROUND OF THE INVENTION

The human hepatitis B virus (HBV) is a member of the hepadnaviridae.Hepadnaviruses are the smallest enveloped DNA viruses which replicatevia reverse transcription of a pgRNA intermediate. During assembly thenucleocapsid acquires three viral envelope proteins termed large (L),middle (M) and small (S). They are encoded in one open reading frame andshare the S-domain which is required for membrane anchoring. In additionto the S-domain, M contains an N-terminal hydrophilic extension of 55amino acids (preS2), while L is further extended by 107, 117 or 118amino acids (genotype-dependent) termed preS1 (Urban 2008). Thehepatitis D virus (HDV) is a satellite virusoid utilizing the HBVenvelope proteins for entry into hepatocytes. The myristoylatedpreS1-domain of L is known to play the key role in HBV and HDVinfectivity.

The inventors have previously identified HBV L-protein derivedlipopeptides that block HBV and HDV infection of PHH and HepaRG cells(Gripon et al., 2005, Schulze et al., 2010, WO 2009/092611 A1). Theyrepresent the N-terminal 47 amino acids of the preS1-domain of HBV(HBVpreS/2-48^(myr)) and include the naturally occurring modificationwith myristic acid.

In WO 2009/092612 and WO 2012/107579, whose contents are incorporatedherewith by reference in its entirety, the inventors describehydrophobic modified preS-derived peptides of HBV and their use asvehicles for the specific delivery of compounds to the liver.

The inventors have furthermore previously identified the receptorresponsible for the binding of these HBV L-protein derived lipopeptides,namely sodium taurocholate co-transporting polypeptide (NTCP/SLC10A1).(U.S. Provisional application 61/725,144, filed Nov. 12, 2012). NTCP isan integral transmembrane protein, not expressed in HepG2, HuH7, inducedin HepaRG cells after DMSO treatment (Kotani et al., 2012) anddown-modulated in primary hepatocytes during de-differentiation (Doringet al., 2012).

In particular, the inventors have identified a novel HBV preS1-specificreceptor playing a key role in Hepatitis B virus (HBV) and/or HepatitisD virus (HDV) infection, the human sodium taurocholate cotransporterpolypeptide NTCP/SLC10A1. Expression of this receptor or of certainnon-human counterparts allows to transform cells that were previouslyunable to bind HBV and/or HDV and/or non-susceptible to HBV and/or HDVinfection into cells that are HBV and/or HDV binding-competent and/orsusceptible to HBV and/or HDV infection. Cells that are alreadysusceptible to HBV and/or HDV infection (HepaRG cells) show asignificantly increased susceptibility upon expression of NTCP.

Also Yan et al. (2012) identified NTCP/SLC10A1 as a preS-specificreceptor in primary Tupaia hepatocytes (PTH) and demonstrate that human(h) NTCP promotes HBV/HDV entry into hepatoma cells.

The liver plays a predominant role in drug biotransformation anddisposition from the body. In view of its barrier function between thegastrointestinal tract and systemic blood, it is constantly exposed toingested xenobiotics entering the portal circulation. Drug-induced liverinjury accounts for up to 7% of all reports of adverse drug effectsvoluntarily reported to pharmacovigilance registries. Drugs cause directdamage to hepatocytes, bile ducts or vascular structures or mayinterfere with bile flow. The phenotypes commonly encountered thusinclude hepatitis, cholestasis, steatosis, cirrhosis, vascular andneoplastic lesions and even fulminant hepatic failure. Almost every drughas the potential to cause hepatic injury, be it through direct toxicityof the agent or through an idiosyncratic response of the individual. Thesusceptibility of the liver to injury by drugs is influenced by variousfactors such as age, sex, pregnancy, comedication, renal function andgenetic factors (Kullak-Ublick, 2000).

Drug induced cholestatic liver disease is a subtype of liver injury thatis characterized by predominant elevations of alkaline phosphatase andbilirubin secondary to the administration of a hepatotoxic agent. It canmanifest itself as a cholestatic hepatitis or as bland cholestasis,depending upon the causative agent and the mechanism of injury. Drugsthat typically cause cholestasis with hepatitis include psychotropicagents, antibiotics and nonsteroidal antiinflammatory drugs (NSAIDs).The mechanism is immunoallergic and results from hypersensitivity. Purecholestasis without hepatitis is observed most frequently withcontraceptive and 17α-alkylated androgenic steroids and the mechanismmost likely involves interference with hepatocyte canalicular effluxsystems for bile salts, organic anions and phospholipids. Therate-limiting step in bile formation is considered to be the bile saltexport pump (BSEP) mediated translocation of bile salts across thecanalicular hepatocyte membrane. Inhibition of BSEP function bymetabolites of cyclosporine A, troglitazone, bosentan, rifampicin andsex steroids is an important cause of drug induced cholestasis(Kullak-Ublick, 2000).

There is a need in the art for improved means and methods for treatingliver involved metabolic diseases, drug induced toxicity and cholestaticliver diseases, as well as cardiovascular diseases.

SUMMARY OF THE INVENTION

According to the present invention this object is solved by providing alipopeptide-based compound for use in the diagnosis, prevention and/ortreatment of a liver disease or condition,

wherein said liver disease or condition is related to sodiumtaurocholate cotransporter polypeptide (NTCP)-mediated transport ofcompounds into hepatocytes.

According to the present invention this object is solved by providing alipopeptide-based compound for use in the diagnosis, prevention and/ortreatment of a cardiovascular disease.

According to the present invention this object is solved by an in vitroor in vivo assay or method for testing or measuring the NTCP-mediatedtransport of test compound(s), comprising the steps of

(a) providing test compound(s) and a lipopeptide-based compound asdefined in the present invention;

(b) providing a test system for functional and selective NTCPexpression;

(c) adding the test compound(s), either together with or without thelipopeptide-based compound, to the NTCP test system of (b);

(d) determining whether the test compound(s) are transported via NTCP bycomparing the results of step (b) and (c) each with or without theaddition of the lipopeptide-based compound,

wherein a test compound is considered being transported via NTCP whenthe compound(s) decrease, block or inhibit bile salt transport by NTCP(competitive transport) or when the transport of the compound(s) can bedecreased, blocked or inhibited by the addition of the lipopeptide-basedcompound.

According to the present invention this object is solved by a method forthe diagnosis, prevention and/or treatment of a liver disease orcondition,

wherein said liver disease or condition is related to sodiumtaurocholate cotransporter polypeptide (NTCP)-mediated transport ofcompounds into hepatocytes, comprising administering a therapeuticallyeffective amount of a lipopeptide-based compound to a patient.

According to the present invention this object is solved by a method forthe diagnosis, prevention and/or treatment of a cardiovascular disease.

According to the present invention this object is solved by a method forthe control or modification of the cholesterol level or cholesteroluptake, comprising administering a therapeutically effective amount of alipopeptide-based compound to a patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E hNTCP specifically binds lipopeptide MyrB.

Stable human NTCP (hNTCP) expression in five hepatoma cell lines wasaccomplished by lentiviral transduction following antibiotic selection.

(A) Western Blots of deglycosylated cell lysates from HuH7^(hNTCP),HepG2^(hNTCP), HepaRG^(hNTCP) Hepa1-6^(hNTCP) and Hep56.1D^(hNTCP) celllines in comparison to mock-transduced cells and two PHH samples. Only10% of sample was loaded on the HepaRG^(hNTCP) lane(*).

(B-C) hNTCP expressing human cell lines were incubated with theAtto488-labeled peptide MyrB^(atto) (green or 488λ). Peptide binding wasanalysed by co-localisation of the peptide with hNTCP-IF using anhNTCP-specific antibody (red) (B) or FACS using the mutant peptideMyrB^(attoAla11-15) or an excess of unlabeled MyrB (C).

(D) FACS analysis of MyrB binding as described in (C) for theHepG2^(mNtcp) cell lines.

(E) HepG2 ratNtcp-eGFP expressing cells (green) were incubated withMyrB^(atto) (red) and analysed by confocal microscopy. Note theco-localisation of hNTCP/MyrB^(atto) in microvilli.

FIGS. 2A-2E Influence of lipopeptide MyrB on NTCP-mediated bile acidtransport; effect of bile acids on HBV infection.

(2A) rNtcp-eGFP expressing HepG2 cells were incubated with increasingconcentrations of MyrB or mutant MyrB^(Ala11-15) (a mutant with Alamutations in the region 9-NPLGFFP-15 (amino acid positions 20-26 of SEQID NO:2), namely 9-NPAAAAA-15 (amino acid positions 8-14 of SEQ IDNO:21)) and ³H-taurocholate uptake was quantified. Uncompeted uptake wasset to 100%.

(2B) hNtcp-eGFP expressing HepG2 cells were incubated with increasingconcentrations of MyrB, mutant MyrB^(Ala11-15) (or preS2-78myr and³H-taurocholate uptake was quantified. Uncompeted uptake was set to100%.

(2C-2D) Differentiated HepaRG (B) or HuH7^(hNTCP) cells (C) werepreincubated 2 h before and coincubated during HBV infection with 5, 50and 500 μM TC, TDC or TCDC and secreted HBeAg was determined d7-9 p.i.Infection was controlled by addition of MyrB 2 h prior to and duringinfection.

(2E) HuH7^(hNTCP) cells were incubated at the indicated bile saltconcentrations overnight at 37° C., trypsinized and incubated in thepresence of bile salts with MyrB^(atto) for further 30 min. Binding wasquantified by FACS analysis. Untagged MyrB was used as a control.

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Before the present invention is described in more detail below, it is tobe understood that this invention is not limited to the particularmethodology, protocols and reagents described herein as these may vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims. Unless defined otherwise, all technical andscientific terms used herein have the same meanings as commonlyunderstood by one of ordinary skill in the art. For the purpose of thepresent invention, all references cited herein are incorporated byreference in their entireties.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “at least 4 amino acids,preferably 4 to 19” should be interpreted to include not only theexplicitly recited values of 4 to 19, but also include individual valuesand sub-ranges within the indicated range. Thus, included in thisnumerical range are individual values such as 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, and sub-ranges such as from 4 to 10,from 6 to 15, from 10 to 19, from 8 to 19 and from 15 to 19, etc. As anillustration, a numerical range of “at least 1 amino acid, preferably 1to 78” should be interpreted to include not only the explicitly recitedvalues of 1 to 78, but also include individual values and sub-rangeswithin the indicated range. Thus, included in this numerical range areindividual values such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 75, 76,77, 78, and sub-ranges such as from 10 to 50, from 15 to 40, from 8 to35, from 30 to 50, and from 20 to 40, etc. This same principle appliesto ranges reciting only one numerical value. Furthermore, such aninterpretation should apply regardless of the breadth of the range orthe characteristics being described.

Use of Lipopeptides in the Treatment of Liver Diseases

As discussed above, the present invention provides a lipopeptide-basedcompound for use in the diagnosis, prevention and/or treatment of aliver disease or condition.

Said liver disease or condition is related to sodium taurocholatecotransporter polypeptide (NTCP)-mediated transport of compounds intohepatocytes.

Preferably, said liver disease or condition that is related toNTCP-mediated transport of compounds into hepatocytes, is a liverinvolved metabolic disease selected from

-   -   intrahepatic cholestasis,    -   poisoning of the liver (by liver toxins)/hepatotoxicity,    -   drug-induced cholestatic liver disease,    -   hyperlipidemia.

Lipopeptide-Based Compound

The lipopeptide-based compound preferably comprises:

-   -   (a) a peptide or amino acid sequence,    -   (b) a hydrophobic or lipid-modification, preferably at the        peptide (a),    -   (c) optionally, a further moiety or further moieties.

Preferably, the peptide or amino acid sequence (a) has or comprises thegeneral formulaX—P—Y

wherein

P is the amino acid sequence NPLGFXaaP SEQ. ID NO: 1,

-   -   (single letter amino acid code)    -   wherein Xaa is an arbitrary amino acid; preferably F or L, more        preferably F    -   (thus, P is preferably NPLGFFP (amino acid positions 20-26 of        SEQ ID NO:2) or NPLGFLP (amino acid positions 8-14 of SEQ ID        NO:21);

X is an amino acid sequence having a length of m amino acids,

-   -   wherein m is at least 4;

Y is an amino sequence having a length of n amino acids,

-   -   wherein n is 0 or at least 1;

and wherein m+n≥11.

The peptide or amino acid sequence (a) (having the general formulaX—P—Y) is preferably derived from the preS domain of hepatitis B virus(HBV) (also designated “preS-peptide”). The envelope of HBV enclosesthree proteins termed L (large), M (middle) and S (small). They sharethe C-terminal S-domain with four transmembrane regions. The M-andL-protein carry additional N-terminal extensions of 55 and,genotype-dependent, 107 or 118 amino acids (preS2- and preS1).

A peptide or amino acid sequence (a) preferably refers to a peptide withan amino acid sequence that corresponds to or is based on the N-terminalextensions of the L-protein of HBV, preS1, preferably of genotypes A toH as well as of woolly monkey (WMHBV), orangutan, chimpanzee and gorillahepatitis B viruses, but it also refers to variants thereof, preferablyC-terminally truncated variants, amino acid substitution variants.

As an indispensable or essential sequence, the amino acid residues beingimportant for the binding of the lipopeptide-based compounds of thepresent invention to NTCP, as set out in SEQ ID NO: 1 (NPLGFXaaP) arepresent in the peptide/amino acid sequence (a) of the lipopeptide-basedcompounds of the invention.

In particular, the peptides are based on the following sequences (aminoacids in single letter code; essential domain underlined).

Essential domain (SEQ ID NO: 1):NPLGFXP (wherein X or Xaa is an arbitrary amino acid, preferablyF or L, more preferably F) preS HBV-A (ID: M57663; SEQ ID NO: 2):MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPIKDHWPQANQVGVGAFGPGFTPPHGGVLGWSPQAQGILATVPAMPPPASTNRQSGRQPTPISPPLRDSHPQApreS HBV-B (ID: D00329, SEQ ID NO: 3)MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDAHKVGVGAFGPGFTPPHGGLLGWSPQAQGILTSVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQApreS HBV-C (ID: AB048704, SEQ ID NO: 4)MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFKANSENPDWDLNPHKDNWPDAHKVGVGAFGPGFTPPHGGLLGWSPQAQGILTSVPAAPPPASTNRQSGRQPTPLSPPLRDTHPQApreS HBV-Chimpanzee (ID: AB032432, SEQ ID NO: 5)MGQNLSTSNPLGFFPEHQLDPAFKANTNNPDWDFNPKKDYWPEANKVGAGAFGPGFTPPHGGLLGWSPQAQGILTTLPANPPPASTNRQSGRQPTPLSPPLRDTHPQApreS HBV-D (ID: AB048702, SEQ ID NO: 6)MGQNLSTSNPLGFFPDHQLDPAFRANTNNPDWDFNPNKDTWPDANKVGAGAFGLGFTPPHGGLLGWSPQAQGFQTLPANPPPASTNRQSGRQPTPLSPPLRTTHPQApreS HBV-E (ID: X65657, SEQ ID NO: 7)MGLSWTVPLEWGKNISTTNPLGFFPDHQLDPAFRANTRNPDWDHNPNKDHWTEANKVGVGAFGPGFTPPHGGLLGWSPQAQGMLKTLPADPPPASTNRQSGRQPTPITPPLRDTHPQApreS HBV-F (ID: X69798@8, SEQ ID NO: 8)MGAPLSTTRRGMGQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDSWPMANKVGVGGYGPGFTPPHGGLLGWSPQAQGVLTTLPAIDPPPASTNRRSGRKPTPVSPPLRDTHPQApreS HBV-G (ID: AF160501, SEQ ID NO: 9)MGLSWTVPLEWGKNLSASNPLGFLPDHQLDPAFRANTNNPDWDFNPKKDPWPEANKVGVGAYGPGFTPPHGGLLGWSPQSQGTLTTLPADPPPASTNRQSGRQPIPISPPLRDSHPQAHBV Gibbon (ID: AJ131572, SEQ ID NO: 10)MGQNHSVINPLGFFPDHQLDPLFRANSNNPDWDFNPNKDTWPEATKVGVGAFGPGETPPHGGLLGWSPQAQGILTTLPAAPPPASTNRQSGRKATPISPPLRDTHPQAHBV-H (ID: Q8JMY6, SEQ ID NO: 11)MGAPLSTARRGMGQNLSVPNPLGFFPDHQLDPLFRANSSSPDWDFNTNKDNWPMANKVGVGGFGPGFTPPHGGLLGWSPQAQGILTTSPPDPPPASTNRRSGRKPTPVSPPLRDTHPQAHBV Orangutan (ID: AF 193864, SEQ ID NO: 12)MGQNLSVSNPLGFFPEHQLDPLFRANTNNPDWDFNPNKDTWPEATKVGVGAFGPGFTPPHGGLLGWSPQAQGVTTILPAVPPPASTNRQSGRQPTPISPPLRDTHPQAHBV Woolly Monkey (ID: NC 001896, SEQ ID NO: 13)MGLNQSTFPLGFFPSHQLDPLFKANAGSADWDKPKDPWPQAHDTAVGAFGPGLVPPHGGLLGWSSQAQGLSVTVPDTPPPPSTNRDKGRKPTPATPPLRDTHPQA

There also exists a HBV preS consensus sequence (for amino acidpositions (−11) to 48) (SEQ ID NO: 14):

(-11)-M GGWSS TPRKG MGTNL SVPNP LGFFP DHQLD PAFRANSNNP DWDFN PNKDH WPEAN KVG-48

“Variants” are preferably N-terminally and/or C-terminally truncatedvariants, amino acid substitution or deletion variants, or prolongedvariants of the sequences of SEQ ID NOs: 2-14, carrying a hydrophobicmodification and wherein, optionally, one or more further moiety ormoieties is/are coupled to one or amino acid(s) N- or C-terminal of theessential domain. Variants comprise furthermore an amino acid sequencecomprising modified amino acid(s), unnatural amino acid(s) orpeptidomimetic(s) or further compounds which can mimic a peptidebackbone/structure. Preferably, variants are selected from C-terminallytruncated variants of SEQ ID NOs. 2 to 14; amino acid substitution ordeletion variants; variants comprising modified amino acid(s), unnaturalamino acid(s) or peptidomimetic(s) or further compounds which can mimica peptide backbone/structure.

Furthermore, the peptide or amino acid sequences are preferably L-aminoacid sequences, but can also comprise D-amino acids or are D-amino acidsequences.

According to the invention, the peptide of the lipopeptide-basedcompound comprises at least the amino acids having the sequence of SEQID NO: 1 and can consist of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118 or 119 amino acids of the above SEQ ID NOs: 2 to 14,or variants thereof.

N-terminally and/or C-terminally truncated variants comprise preferablyat least 18 consecutive amino acids, more preferably at least 19consecutive amino acids, even more preferably at least 20 and just evenmore preferably at least 21 consecutive amino acids of SEQ ID NOs. 2 to14 or variants thereof.

The N-terminal sequence X of the peptide having a length of m aminoacids comprises at least 4 amino acids (i.e. m is at least 4).Preferably, the N-terminal sequence X can consist of 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids. That is, m may be4 to 19.

In one embodiment, one or amino acid(s) of X have an amino group in aside chain, which is/are preferably selected from lysine, α-aminoglycine, α,γ-diaminobutyric acid, ornithine, α,β-diaminopropionic acid,more preferably lysine. The amino acid(s) of X having an amino group ina side chain, is/are preferably is/are located at the N-terminus of X,wherein one to eleven (1-11), preferably one to three (1-3), amino acidshaving an amino group in a side chain are located at the N-terminus ofX.

In one embodiment, the N-terminal sequence X preferably comprises thesequence NX₁SX₂X₃ (SEQ ID NO: 15), wherein X₁, X₂ and, X₃ may bearbitrary amino acids. Preferably, X₁ of SEQ ID NO: 15 is L, I or Q,more preferably L. Preferably, X₂ of SEQ ID NO: 15 is T, V, A or is notpresent, preferably T or V, more preferably T. Preferably, X₃ of SEQ IDNO: 15 is P, S, T or F, more preferably P or S, even more preferably S.Preferably, the sequence NX₁SX₂X₃ (SEQ ID NO: 15) is directly attachedto the N-terminus of the amino acid sequence P (SEQ. ID NO: 1;NPLGFXaaP), resulting in a peptide comprising the sequenceNX₁SX₂X₃NPLGFXaaP, wherein X₁, X₂, X₃ and Xaa are defined as above.

The C-terminal sequence Y of the peptide having a length of n aminoacids comprises 0 or at least 1 amino acids (i.e. n=0 or n is at least1). Preferably, the C-terminal sequence Y can consist of 0, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51. 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 or 93 aminoacids. That is, n may be 0 to 93.

In one embodiment, the C-terminal sequence Y consists of at least 4amino acids (i.e. n is at least 4), which preferably has the sequenceX₄HQLDP (SEQ ID NO: 16), wherein X₄ is an arbitrary amino acid.Preferably, X₄ of SEQ ID NO: 16 is D, E or S, more preferably D or E,even more preferably D. Preferably, the sequence X₄HQLDP (SEQ ID NO: 16)is directly attached to the C-terminus of the amino acid sequence P(SEQ. ID NO: 1; NPLGFXaaP), resulting in a peptide comprising thesequence NPLGFXaaPX₄HQLDP, wherein X₄ and Xaa are defined as above.

In a preferred embodiment, the peptide of the lipopeptide-based compoundof the present invention comprises a peptide encoded by the amino acidsequence NX₁ SX₂X₃NPLGFXaaP X₄HQLDP (SEQ ID NO: 17), wherein X₁, X₂, X₃,X₄ and Xaa are defined as above.

The term “variant” also refers to the homologous sequences found in thedifferent viral species, strains or subtypes of the hepadnavirus genus,such as HBV strain alpha, HBV strain LSH (chimpanzee isolate), woollymonkey HBV (WMHBV), or strains selected from the group consisting of theHBV genotypes A to H (see SEQ ID NO: 2-13).

The term “variant” also refers to homologous sequences which show atleast 50% sequence identity to an amino acid sequence comprising theinvariant NPLGFXaaP-domain and the adjacent sequences of SEQ ID NO: 2-14or any other amino acid sequence disclosed herein, preferably 70%, morepreferably 80%, even more preferably 90% or 95%.

Thus, a preferred peptide/amino acid sequence (a) according to theinvention comprises a variant of SEQ ID NOs: 2 to 14 with an amino acidsequence of the different viral species, strains or subtypes, preferablyof the genotypes of HBV or woolly monkey HBV (WMHBV) or variantsthereof.

“Variants” of SEQ ID NOS: 2 to 14 also comprise variants or “analogues”comprising amino acid deletions, amino acid substitutions, such asconservative or non-conservative replacement by other amino acids or byisosteres (modified amino acids that bear close structural and spatialsimilarity to protein amino acids), amino acid additions or isostereadditions, as long as the sequence still binds to NTCP.

Conservative amino acid substitutions typically relate to substitutionsamong amino acids of the same class. These classes include, for example,

amino acids having uncharged polar side chains, such as asparagine,glutamine, serine, threonine and tyrosine;

amino acids having basic side chains, such as lysine, arginine, andhistidine;

amino acids having acidic side chains, such as aspartic acid andglutamic acid; and

amino acids having nonpolar side chains, such as glycine, alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and cysteine.

As discussed above, the peptide or amino acid sequences (a) arepreferably L-amino acid sequences, but can also comprise D-amino acidsor are D-amino acid sequences.

Preferably, the peptide or amino acid sequence (a), X—P—Y is selectedfrom a peptide comprising an amino acid sequence selected from

SEQ ID NO: 18 HBV preS/2-48 (genotype C), SEQ ID NO: 19 HBV preS/2-48(genotype D), SEQ ID NO: 20 HBV preS/2-48 (consensus),or

-   -   an amino acid sequence having at least 90% sequence identity        (preferably at least 95% or 99% identity) to the above sequences    -   and variants thereof.

In this embodiment, m=7 and n=33 (m+n=40), resulting in a peptide oramino acid sequence of 47 amino acids.

SEQ ID NO: 18 GTNL SVPNP LGFFP DHQLD PAFGA NSNNP DWDFN PNKDH WPEAN KVGSEQ ID NO: 19 GQNL STSNP LGFFP DHQLD PAFRA NTANP DWDFN PNKDT WPDAN KVGSEQ ID NO: 20 GINL SVPNP LGFFP DHQLD PAFRA NSNNP DWDFN PNKDH WPEAN KVG

In a preferred embodiment, the lipopeptide is Myrcludex B:

(a) having the amino acid sequence of HBV preS/2-48 (genotype C) withSEQ ID NO: 18.

The lipopeptide-based compound according to the invention preferablycomprises:

(b) A Hydrophobic or Lipid-Modification, Preferably at the Peptide (a).

Preferably, the peptide comprises a hydrophobic or lipid-modification(b), such as at the N-terminus, the C-terminus or at an amino acid sidechain.

In one embodiment, the peptide is modified with at least one hydrophobicmoiety or group. In preferred embodiments of this invention, the peptideis modified with 1, 2, 3, 4 or more hydrophobic moiety/ies or group(s).That is, the peptide can be modified with more than one hydrophobicmoiety or group, such as 2. The hydrophobic moieties or groups can bethe same or different to each other.

The hydrophobic modification is preferably selected from: acylationand/or addition of hydrophobic moieties.

Preferably, the peptide comprises an N or C-terminal hydrophobicmodification (b).

An N-terminal hydrophobic modification is preferred.

“N-terminal” refers to the N-terminus of a peptide, thus in a peptidewith the general formula X—P—Y, it refers to the N-terminus of X, i.e.the respective first amino acid residue, but comprises also thehydrophobic modification in close proximity to the N-terminus, such asrespective amino acid residues (−4), (−3), (−2), (−1), 1, 2 or 3 or 4.Thus, the coupling of the hydrophobic modification can furthermore beobtained by an attachment of a hydrophobic moiety at a site close to theN-terminus of X.

The hydrophobic modification of the lipopeptide-based compound accordingto the present invention adds a hydrophobic moiety, preferably to thepeptide/amino acid sequence.

Acylation is preferably selected from acylation with carboxylic acids,fatty acids, amino acids with lipophilic side chains. Preferred fattyacids are saturated or unsaturated fatty acids, branched or unbranchedfatty acids, preferably with 8 to 22 carbon atoms (C 8 to C 22). Morepreferably, the hydrophobic modification by acylation is selected fromacylation with myristoyl (C 14), palmitoyl (C 16) or stearoyl (C 18).Modification by myristoylation is preferred in in vivo and medicinalapplications due to its higher safety, e.g. not showing the adverseeffects of the stearoyl group (innate immune response etc).

The addition of hydrophobic moieties is preferably selected fromaddition of cholesterol, derivatives of cholesterol, phospholipids,glycolipids, glycerol esters, steroids, ceramids, isoprene derivatives,adamantane, farnesol, aliphatic groups, polyaromatic compounds, oleicacid, bile salts or bile salt conjugates, more preferably oleic acid,cholesterol, bile salts or bile salt conjugates. The attachment of thehydrophobic moieties is preferably by covalent binding, which can beachieved via carbamate, amide, ether, disulfide or any other linkagethat is within the skill of the person skilled in the art.

Thus, the peptide/amino acid sequences (a) are preferablyhydrophobically modified, preferably acylated and, thus, preferablylipopeptides due to their lipophilic or hydrophobic group/moiety.

In one embodiment, the peptide or amino acid sequence (a) has orcomprises the general formulaX—P—Y—R_(o)

wherein P, X, and Y are as defined above and

R is a C-terminal modification of said hydrophobic modified peptide,

-   -   which is preferably a moiety that protects from degradation        selected from amide, D-amino acid, modified amino acid, cyclic        amino acid, albumin, natural and synthetic polymer, such as PEG,        glycane,    -   o is 0 or at least 1.

The C-terminal modification (R) of Y is preferably a modification with amoiety that protects from degradation, such as in vivo degradation.

“C-terminal” refers to the modification at the C-terminus, i.e. therespective last amino acid residue, but comprises also the modificationin close proximity to the C-terminus, such as the last but one aminoacid residue, the last but two amino acid residue or more amino acidresidues (e.g. introduction of one D-amino acid that protects thecarrier from enzymatic degradation e.g. by the action ofcarboxypeptidases). The skilled artisan will be able to select therespective suitable moiety(s) depending on the respective application.Preferred moieties that protect from degradation are selected fromamides, D-amino acids, modified amino acids, cyclic amino acids,albumin, natural and synthetic polymers, such as PEG, glycane.

Furthermore, o is 0 or at least 1, i.e. the C-terminal modification (R)is optional. Preferably, o is 1. In further embodiments of thisinvention o is 1, 2, 3, 4 or more. That is, the C-terminus or itsproximity can be modified with more than one moiety or group, such as 2.The moieties or groups can be the same or different to each other.

In one embodiment, the preferred C-terminal modification is an amide.

In an embodiment of this invention the hydrophobic modification and/or Rare linked to the peptide via a linker or spacer. Linker or spacer areknown to the skilled artisan, such as polyalanine, polyglycin,carbohydrates, (CHa)n groups. The skilled artisan will, thus, be able toselect the respective suitable linker(s) or spacer(s) depending on therespective application.

In a preferred embodiment, the lipopeptide is Myrcludex B having

(a) the amino acid sequence of HBV preS/2-48 (genotype C) with SEQ IDNO. 18;

(b) an N-terminal myristoylation

(c) a C-terminal amide.

Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-amide

In one embodiment, the lipopeptide-based compound according to theinvention comprises

(c) A Further Moiety or Moieties.

Such further moieties can be

-   -   drug(s) or their respective prodrug(s);    -   tag(s);    -   label(s), such as fluorescent dye(s), radioisotope(s) and        contrast agent(s);    -   recombinant virus(s) or derivative(s) thereof;    -   carrier or depot(s) for drug(s), prodrug(s) or label(s);    -   immunogenic epitope(s);    -   hormones (peptide hormones, steroid hormones, monoamines, amino        acid derivatives, eicosanoids).

In one embodiment, the further moiety or moieties are covalentlyattached to the lipopeptide-compound (preferably to the peptide), suchas via linker, spacer and/or anchor group(s).

The lipopeptide-based compounds can further contain anchor group(s) thatcan serve as an additional point(s) of attachment for further moieties(such as compound, tag, label) and can be located at an amino acid of Y.

An anchor group can be at an amino acid side chain or can be the aminoacid side chain itself, i.e. the anchor group can be a side chain itselfor a modified side chain. The anchor group can also be a modified aminoacid residue which was introduced into the amino acid sequence of thelipopeptide to serve as an anchor group. In other embodiments of theinvention the anchor group A is attached to the hydrophobic modificationand/or the C-terminal modification R.

Preferred anchor groups are selected from ester, ether, disulfide,amide, thiol, thioester. The skilled artisan will be able to select therespective suitable anchor group(s) depending on the respective furthermoiety to be attached. The anchor group can furthermore be suitable forattaching a complex-forming component, such as of the biotin/avidin,polyarginine/oligonucleotide (e.g. siRNA) complex. In some embodiments,there are more than one anchor group, such as 2, 3, 4 or more, such as2. The anchor groups can be the same or different to each other,allowing the attachment of several further moieties.

In one embodiment, the further moiety/moieties is/are contrast agent(s)which are coupled via a chelating agent.

Thereby, the contrast agent is bound/coupled in the form of a complexwith a chelating agent being able to form complexes with the respectivecontrast agent.

Such chelating agent can be1,4,7,10-tetraazacyclododecane-N,N′,N,N′-tetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA),1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA),triethylenetetramine (TETA), iminodiacetic acid,Diethylenetriamine-N,N,N′,N,N″-pentaacetic acid (DTP A) and6-Hydrazinopyridine-3-carboxylic acid (HYNIC), such as preferably DOTA.

Examples of contrast agents are paramagnetic agents, e.g. Gd, Eu, W andMn, preferably complexed with a chelating agent. Further options aresupramagnetic iron (Fe) complexes and particles, compounds containingatoms of high atomic number, i.e. iodine for computer tomography (CT),microbubbles (such as for contrast enhanced ultrasound (CEUS)) andcarriers such as liposomes that contain these contrast agents.

The peptides of the invention can be prepared by a variety of proceduresreadily known to those skilled in the art, in general by syntheticchemical procedures and/or genetic engineering procedures. Syntheticchemical procedures include more particularly the solid phase sequentialand block synthesis. More details can be taken from WO 2009/092612.

NTCP

Sodium/bile acid cotransporter also known as the sodium/Na⁺-taurocholatecotransporting polypeptide (NTCP) is a protein that in humans is encodedby the SLC10A1 (solute carrier family 10 member 1) gene.

Sodium/bile acid cotransporters are integral membrane glycoproteins thatparticipate in the enterohepatic circulation of bile acids. Twohomologous transporters are involved in the reabsorption of bile acids,one absorbing from the intestinal lumen, the bile duct, and the kidneywith an apical localization (SLC10A2), and the other sodium-dependentcotransporter being found in the basolateral membranes of hepatocytes(SLC10A1).

Bile formation is an important function of the liver. Bile salts are amajor constituent of bile and are secreted by hepatocytes into bile anddelivered into the small intestine, where they assist in fat digestion.In the liver, hepatocytes take up bile salts (mainly via NTCP) andsecrete them again into bile (mainly via the bile salt export pump(BSEP)) for ongoing enterohepatic circulation. Uptake of bile salts intohepatocytes occurs largely in a sodium-dependent manner by the sodiumtaurocholate cotransporting polypeptide NTCP. The transport propertiesof NTCP have been extensively characterized. It is an electrogenicmember of the solute carrier family of transporters (SLC10A1) andtransports predominantly bile salts and sulfated compounds, but is alsoable to mediate transport of additional substrates, such as thyroidhormones, drugs and toxins. It is highly regulated under physiologic andpathophysiologic conditions. Regulation of NTCP copes with changes ofbile salt load to hepatocytes and prevents entry of cytotoxic amounts ofbile salts during liver disease.

For a review of bile salt transporters, see also Trauner and Boyer(2003).

For NTCP a large range of substrates could be detected, it transportsunconjugated as well as taurine-conjugated and glycine-conjugated bileacids (Hagenbuch & Meier, 1994), also sulfated bile acids and, incontrast to the apical sodium dependent bile acid transporter (ASBT),also steroid sulfates (Craddock et al 1998; Kramer et al, 1999;Schroeder et al 1998), and thyroid hormones (Friesema et al, 1999).Drugs like rosuvastatin (Ho et al., 2006) and micafungin (Yanni et al.,2010) have also been shown to have affinity for NTCP. Recent data showFDA-approved drugs that are identified as inhibitors of NTCP (Dong etal., 2013). Most of them are antifungal, antihyperlipidemic(simvastatin), antihypertensive, anti-inflammatory, or glucocorticoiddrugs.

Preferably, the compounds which are transported into hepatocytes viaNTCP are

-   -   bile acids        -   such as cholate        -   taurine- or glycine conjugated bile acids and salts thereof            -   (taurine- or glycine conjugated dihydroxy and trihydroxy                bile salts)            -   such as            -   taurocholate            -   glycocholate            -   taurodeoxycholate            -   taurochenodeoxycholate            -   tauroursodeoxycholate        -   sulfated bile acids and salts thereof    -   steroides        -   steroide sulfates            -   estrogen conjugates (e.g. estrone-3-sulfate,                17α-ethinylestradiol-3-O-sulfate)            -   dehydroepiandrosterone sulfate    -   conjugated and non-conjugated thyroid hormones    -   liver toxins    -   compounds that are covalently bound to taurocholate (e.g.        chlorambucil-taurocholate)    -   bromosulphophthalein,    -   drugs        -   such as        -   antifungal (e.g. micafungin),        -   antihyperlipidemic (e.g. simvastatin, rosuvastatin,            pitavastatin, fluvastatin, atorvastatin),        -   antihypertensive,        -   anti-inflammatory, or        -   glucocorticoid drugs.

Preferably, said liver disease or condition that is related toNTCP-mediated transport of compounds into hepatocytes, is a liverinvolved metabolic disease selected from

-   -   intrahepatic cholestasis,    -   poisoning of the liver (by liver toxins)/hepatotoxicity,    -   drug-induced cholestatic liver disease,    -   hyperlipidemia,    -   posthepatic cholestasis.

A “liver involved metabolic disease” when used herein refers tometabolic disorders including visceral obesity, diabetes mellitus anddyslipidemia which are influenced by the liver metabolism of lipids andbile acids.

In general, “cholestasis” is a condition where bile constituents cannotbe secreted from hepatocytes into the biliary tree or where bile cannotflow from the liver to the duodenum, resulting in hepatocyte bile acidaccumulation within hepatocytes.

“Cholestasis” or “intrahepatic cholestasis” when used herein refers tointrahepatic toxic effects of hepatocyte bile acid accumulation relatedto an insufficient expression and/or activity of bile salt pumps (likeBSEP or MRP) in the canalicular membrane.

“Posthepatic cholestasis” when used herein refers to a cholestatic liverdisease due to obstruction of the large bile ducts.

“Poisoning of the liver” or “hepatotoxicity” or “toxic liver disease”when used herein refer to toxic effects of drugs independent of bileacid accumulation. These drugs penetrate the hepatocytes via theNTCP-mediated transport and cause several direct toxic effects, bydamaging the mitochondria or by activating enzymes in the cytochromeP-450 system leading to oxidative stress.

“Drug-induced cholestatic liver disease” when used herein refers toinhibition of the export of bile acids from hepatocytes due to drugeffects on bile salt export pump (BSEP).

Drug-induced cholestasis may be caused by several drugs which inhibitBSEP, such as rifampicin, cyclosporine A, rifamycin SV, bosentan,troglitazone, erythromycin estolate, and glibenclamide (Fattinger etal., 2001; Funk et al., 2001; Funk et al., 2001; Stieger et al., 2000;Dawnson et al., 2012; Morgan et al., 2010; Ogimura et al., 2011). BSEPis a member of the ATP-binding cassette (ABC) family of transporters(BSEP is also identified as ABCB11) and it is involved in the process ofexporting bile acids out of hepatocytes, thus reducing their toxicity tothese cells. The above mentioned drugs cause the toxic effects of excessbile acid accumulation because the excretion of bile acid via BSEP isdisabled. Inhibition of NTCP-mediated bile acid uptake via thelipopeptide-based compound (such as MyrB) and NTCP counterbalances BSEPinhibition, and thereby prevents hepatotoxicity or is suitable fortreatment and/or diagnosis.

“Hyperlipidemia” (or hyperlipoproteinemia, or hyperlipidemia) involvesabnormally elevated levels of any or all lipids and/or lipoproteins inthe blood.

Hyperlipidemias are divided in primary and secondary subtypes. Primaryhyperlipidemia is usually due to genetic causes (such as a mutation in areceptor protein), while secondary hyperlipidemia arises due to otherunderlying causes such as diabetes. Lipid and lipoprotein abnormalitiesare common in the general population, and are regarded as a modifiablerisk factor for cardiovascular disease due to their influence onatherosclerosis.

“Hypercholesterolemia” (or hypercholesterolaemia) is the presence ofhigh levels of cholesterol in the blood. It is a form of“hyperlipidemia”.

“Hyperlipidemia” when used herein preferably refers tohypercholesterolemia which includes elevated LDL cholesterol, reducedHDL cholesterol, elevated triglycerides, clogged arteries leading tohigh blood pressure, cardiovascular disease (CVD), heart attacks andstrokes.

Preferably, the NTCP-mediated transport is decreased or blocked by thelipopeptide-based compound.

The inventors have found that the lipopeptide MyrB interferes withNTCP-mediated bile salt transport. In particular, MyrB inhibitsNTCP-mediated bile salt transport.

Thereby, the K_(i) for transporter inactivation (K_(i) for rNTCP ˜4 nM)is much higher compared to the IC₅₀ observed for HBV/HDV infectioninhibition (80 pM), which coincides with the finding that HBV infectioncan already been blocked at concentrations below receptor saturation(Schulze et al., 2010). A plausible explanation is the assumption thatsimilar to other viruses the L-protein/hNTCP complex has to multimerize.Binding of MyrB to a single subunit could abrogate virus entry whereassubstrate transport may continue. This assumption is supported byreports demonstrating oligomerization of NTCP (Doring et al., 2012).

Preferably, the lipopeptide-based compound is administered in atherapeutically effective amount.

A “therapeutically effective amount” of a lipopeptide-based compound ofthis invention refers to the amount that is sufficient to block orinhibit the NTCP-mediated bile salt transport.

A “therapeutically effective amount” of a lipopeptide-based compound ofthis invention further refers to the amount that is sufficient todiagnose, prevent and/or treat the respective liver disease or disorder.The preferred therapeutically effective amount depends on the respectivecompound that is to be delivered and its respective therapeuticpotential.

The lipopeptide-based compound is preferably used in a concentrationsuch that a K_(i) of about 1 to 10 nM is reached at the target site,i.e. NTCP site (hepatocytes).

In particular, in order to inhibit substrate transport thelipopeptide-based compound is preferably used in a dose such that theconcentration at the target site is above the K_(i) of about 1 to 10 nM.

In case of an IC₅₀ value of the lipopeptide-based compound used of about10 nM, a preferred therapeutically effective amount is about 100 μg perkg body weight or in the range of 1 to 5 mg per patient. The preferredtherapeutically effective amount in the range of 1 to 5 mg per patientcan be administered once a day or in other embodiments only once every2-3 days, depending on stability and metabolism of the compound used andthe turnover of the complex of NTCP/compound.

A therapeutically effective amount is preferably a daily dosage or adaily administration in the range of

from about 0.1 mg to about 50 mg per patient, i.e. from about 0.0014mg/kg body weight to about 0.7 mg/kg body weight,

preferably from about 1 mg to about 20 mg per patient, i.e. from about0.014 mg/kg body weight to about 0.28 mg/kg body weight.

The skilled artisan will be able to determine suitable therapeuticallyeffective amounts.

Preferably, the route of administration or application of the presentinvention is selected from subcutaneous, intravenous, oral, nasal,intramuscular, transdermal, inhalative, by suppository.

A preferred embodiment for nasal administration or application is anasal spray.

In one embodiment, the lipopeptide-based compound of the presentinvention is dissolved in serum from the patient and is applied viainjection.

The preferred therapeutically effective amount depends on the respectiveapplication and desired outcome of inhibition, diagnosis, preventionand/or treatment.

The lipopeptide-based compounds can be administered/applied in form ofpharmaceutical compositions comprising:

at least one lipopeptide-based compound as defined herein above; and

optionally a pharmaceutically acceptable carrier and/or excipient.

Such pharmaceutical compositions are very well suited for all the usesand methods described herein.

A “pharmaceutically acceptable carrier or excipient” refers to anyvehicle wherein or with which the pharmaceutical compositions may beformulated. It includes a saline solution such as phosphate buffersaline. In general, a diluent or carrier is selected on the basis of themode and route of administration, and standard pharmaceutical practice.

Lipopeptides for Use in the Control of the Cholesterol Level and inCardiovascular Diseases

As discussed above, the present invention provides a lipopeptide-basedcompound for use in the control or modification of the cholesterol levelor cholesterol uptake.

The cholesterol level or uptake is controlled or modified by decreasingor blocking the NCTP-mediated bile salt transport by thelipopeptide-based compound as defined in this application.

As discussed above, the present invention provides a lipopeptide-basedcompound for use in the diagnosis, prevention and/or treatment of acardiovascular disease (CVD).

Said uses comprises the control or modification of the cholesterol levelor cholesterol uptake, wherein the cholesterol level or uptake iscontrolled or modified by decreasing or blocking the NCTP-mediated bilesalt transport by the lipopeptide-based compound as defined in thisapplication.

Cardiovascular diseases (CVD) are the major cause of morbidity and deathin the western world. High levels of cholesterol have been associatedwith CVD as one of the rise factors. Of particular importance clinicallyis the abnormal deposition of cholesterol and cholesterol-richlipoproteins in the coronary arteries. Such deposition, eventuallyleading to atherosclerosis, is the leading contributory factor indiseases of the coronary arteries. In this case the management of CVD iscritical dependent on lipid-lowering therapies. Different classes ofdrugs are available for this purpose, such as statins, cholesterolabsorption inhibitors, bile acid resins, fibrates and nicotinic acidthat act by reducing the levels of cholesterol by distinct pathways(Schmitz & Langmann, 2006). These drugs have several side effects anddepend on the relative levels of the metabolizing enzymes andtransporters that act on cardiovascular drugs.

The main control of cholesterol metabolism is caused by bile acid as animportant regulator of cholesterol homeostasis. The levels of bile acidand cholesterol are linked by the regulation of cholesterol metabolismand absorption. The synthesis of the bile acids is the major pathway ofcholesterol catabolism in mammals, because the end products ofcholesterol utilization are the bile acids. The major pathway for thesynthesis of the bile acids is initiated via hydroxylation ofcholesterol at the 7 position via the action of cholesterol7α-hydroxylase (CYP7A1).

That means that the synthesis of bile acids is one of the predominantmechanisms for the excretion of excess cholesterol. Under physiologicalconditions this regulation is insufficient to compensate for an excessintake of cholesterol. However, if bile acid uptake into hepatocytes isblocked, the excretion of cholesterol in the form of bile acids will besufficient to compensate for an excess dietary intake of cholesterol.Blocking bile acid uptake via the lipopeptide-based compound accordingto the invention and NTCP leads to intracellular deficiency of bile acidwhich is compensated by increased cholesterol metabolism and absorption.

Thus, according to the invention, the lipopeptide-based compounds aresuitable for lipid-lowering therapies to prevent CVD.

Assay for NTCP-Mediated Transport of Test Compound(s)

As discussed above, the present invention provides an in vitro and invivo assay or method for testing or measuring the NTCP-mediatedtransport of test compound(s).

Said in vitro and in vivo assay or method comprises the steps of

(a) providing test compound(s) and a lipopeptide-based compound asdefined herein;

(b) providing a test system for functional and selective NTCPexpression, which includes measurement of bile acid transport by NTCP;

(c) adding the test compound(s), either together with or without thelipopeptide-based compound, to the NTCP test system of (b);

(d) determining whether the test compound(s) are transported via NTCP bycomparing the results of step (b) and (c) each with or without theaddition of the lipopeptide-based compound,

wherein a test compound is considered being transported via NTCP whenthe compound(s) decrease, block or inhibit bile salt transport by NTCP(competitive transport) or when the transport of the compound(s) can bedecreased, blocked or inhibited by the addition of the lipopeptide-basedcompound.

The skilled artisan will be able to determine and apply a suitable testsystem or test model. Such a suitable test system comprises thefunctional and selective NTCP expression and thus a functional NTCPtransport, which can selectively be blocked/inhibited by alipopeptide-based compound of the invention (such as MyrB). It can beone or more of the following

a transgenic cell line expressing a functional NTCP,

an transgenic animal expressing a functional NTCP.

Examples for suitable in vitro test systems or test models are:

hepatocyte and hepatoma cell lines stably transduced with anNTCP-encoding lentivirus, as described in the examples and as describedin the U.S. Provisional application 61/725,144 filed Nov. 12, 2012.

Examples for suitable in vivo test systems or test models are:

isolated perfused liver, e.g. mouse or rat, as described in vom Dahl etal., 1991 or Schulz et al., 1991;

transgenic mouse, as described in the example and as described in theU.S. Provisional application 61/725,144 filed Nov. 12, 2012.

Methods for the Treatment of Liver Diseases

As discussed above, the present invention provides a method for thediagnosis, prevention and/or treatment of a liver disease or condition.

Said liver disease or condition is related to sodium taurocholatecotransporting polypeptide (NTCP)-mediated transport of compounds intohepatocytes.

The method of the invention comprises the step of administering atherapeutically effective amount of a lipopeptide-based compound to apatient.

The lipopeptide-based compound is preferably as defined in thisapplication.

Preferably, and as discussed above, said liver disease or condition thatis related to NTCP-mediated transport of compounds into hepatocytes, isa liver involved metabolic disease selected from

intrahepatic cholestasis

poisoning of the liver (by liver toxins)/hepatotoxicity,

drug-induced cholestatic liver disease,

hyperlipidemia,

posthepatic cholestasis.

Preferably, and as discussed above, the compounds which are transportedinto hepatocytes via NTCP are

-   -   bile acids        -   such as cholate        -   taurine- or glycine conjugated bile acids and salts thereof            -   (taurine- or glycine conjugated dihydroxy and trihydroxy                bile salts)            -   such as            -   taurocholate            -   glycocholate            -   taurodeoxycholate            -   taurochenodeoxycholate            -   tauroursodeoxycholate        -   sulfated bile acids and salts thereof    -   steroides        -   steroide sulfates            -   estrogen conjugates (e.g. estrone-3-sulfate,                17α-ethinylestradiol-3-O-sulfate)            -   dehydroepiandrosterone sulfate    -   conjugated and non-conjugated thyroid hormones    -   liver toxins    -   compounds that are covalently bound to taurocholate (e.g.        chlorambucil-taurocholate)    -   bromosulphophthalein,    -   drugs    -   such as        -   antifungal (e.g. micafungin),        -   antihyperlipidemic (e.g. simvastatin, rosuvastatin,            pitavastatin, fluvastatin, atorvastatin),        -   antihypertensive,        -   anti-inflammatory, or        -   glucocorticoid drugs.

In one embodiment, as discussed above, the NCTP-mediated transport isdecreased or blocked by the lipopeptide-based compound.

Preferably, and as discussed above, the therapeutically effective amountof the lipopeptide-based compound is in the range of from about 0.1 mgto about 50 mg per patient and per day, preferably from about 1 mg toabout 20 mg per patient per day.

Methods for Controlling the Cholesterol Level and Treatment ofCardiovascular Diseases

As discussed above, the present invention provides a method for thecontrol or modification of the cholesterol level or cholesterol uptake.

The cholesterol level or uptake is controlled or modified by decreasingor blocking the NCTP-mediated bile salt transport (by thelipopeptide-based compound).

The method of the invention comprises the step of administering atherapeutically effective amount of a lipopeptide-based compound to apatient.

The lipopeptide-based compound is preferably as defined in thisapplication.

As discussed above, the present invention provides a method for thediagnosis, prevention and/or treatment of a cardiovascular disease(CVD), comprising administering a therapeutically effective amount of alipopeptide-based compound, preferably as defined in any of claims 5 to9, to a patient.

Thereby, the cholesterol level or uptake is preferably controlled ormodified by decreasing or blocking the NCTP-mediated bile salt transportby the lipopeptide-based compound as defined in this application.

NTCP-mediated blocking bile acid uptake enables to an elevatedcholesterol turn over via hepatocytes. Hence LDL cholesterol will bereduced and HDL cholesterol will be elevated. As a consequence the riskof clogged arteries leading to high blood pressure, CVD heart attacksand strokes will be minimized.

Further Description of the Invention

The inventors show that the lipopeptide Myrcludex B (MyrB) interfereswith NTCP-mediated bile salt transport.

NlyrB: Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-amide

Functional analyses of the NTCP/SLC10A receptor revealed that:

(i) human NTCP (hNTCP) binds MyrB;

(ii) NTCP-substrates interfere with HBV infection;

(iii) MyrB inhibits NTCP-mediated bile salt transport.

MyrB is an interesting novel drug to target NTCP, but also to study itsfunction in vivo.

Remarkably, the K_(i) for transporter inactivation (K_(i) for rNTCP˜4nM) is much higher compared to the IC₅₀ observed for HBV/HDV infectioninhibition (80 pM) (Schulze et al., 2010). This coincides with thefinding that HBV infection can already been blocked at concentrationsbelow receptor saturation (Schulze et al., 2010). A plausibleexplanation is the assumption that similar to other viruses theL-protein/hNTCP complex has to multimerize. If only one subunit boundMyrB, entry may be abrogated although substrate transport may progress.This assumption is supported by reports demonstrating oligomerization ofNTCP (Doring et al., 2012).The observation that natural substrates ofNTCP, when applied at high concentrations (FIGS. 2C and D) interferewith MyrB binding and HBV infection indicate that sodium driventransport is coupled to effective HBV entry.

The following examples and drawings illustrate the present inventionwithout, however, limiting the same thereto.

EXAMPLES

1. Methods

1.1 Plasmids: hNTCP cDNA (Origene, USA) and mNtcp cDNA (Rose et al.,2011) were subcloned into the puromycin co-expressing lentiviral vectorpWPI-puro. hNTCP, mNtcp and h/mNtcp chimera were generated byoverlapping PCR and introduced into pWPI-GFP.

1.2 Cells: Lentiviruses were produced and used to transduce hNTCP intohuman (HepaRG, HepG2, HuH7), mouse (Hepa1-6, Hep56.1D) and the rathepatoma cell line TC5123. The respective mock transduced cells wereused as controls. To generate stable cell lines, selection with 2.5μg/ml puromycin was achieved. Differentiation of transduced HepaRG wasinduced by DMSO as described (Gripon et al., 2002). HepG2-rNTCP and

HepG2-rNTCP-eGFP cell line have been described previously for expressionof rat Ntcp with or without fused eGFP (Stross et al., 2010).

1.3 Synthesis and Labeling of Peptides

Synthesis of MyrB and the MyrB mutant and the control peptidepreS2-78myr was performed by solid phase synthesis (Schieck et al.,2013). Labelling was achieved by couplingatto565-NHS-ester/atto488-NHS-ester (ATTO-TEC, Germany) to the lysineresidues of the peptides. Monolabelled peptides were pooled after HPLCpurification and stock solutions (100 μM) were prepared and stored at−80° C.

MyrB SEQ ID NO: 18 Myr-GTNLSVPNPLGFFPDHQLDPAFGANSNNPDWDFNPNKDHWPEANKVG-amide mutant MyrB^(Ala11-15) SEQ ID NO: 21 Myr-GTNLSVPNPAAAAADHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-amide preS2-78myr SEQ ID NO: 22 Myr-ggnlstsnplgffpdhqldpafrantanpdwdfnpnkdtwpdankvgagafglgftpphggllgwsidgaggilqtlp

1.4 Flow Cytometry: Cells were incubated for 30 min at 37° C. in mediumcontaining 200 nM MyrB^(atto) or the MyrB^(atto)-mutant. Cells werewashed (PBS/1% BSA), trypsinized, and suspended inKrebs-Henseleit-Buffer. Flow cytometry was performed on a FACS Canto II(BD Bioscience, Heidelberg, Germany); FlowJo v7.61 software (Treestar,Ashton, USA) was used for analysis. Compensation was performed using BDCompbeats (BD Bioscience, Heidelberg, Germany).

1.5 IF: Cells were grown on coverslips (see Meier et al., 2012) washedand incubated with 400 nM MyrB (37° C.; 30 min). Cells were washed again(3× PBS/2% BSA), fixed with PFA, washed with PBS/1 ug/ml Hoechst 3342and mounted (FluoromountG). NTCP immune staining was achieved afterpermeabilisation (10 min/RT) with TritonX 100 using a α-SCL10A1/NTCPantibody (Sigma, Germany) diluted 1:750 in PBS/2% BSA (18 h at 4° C.). Apolyclonal rabbit antiserum H863 was used for HBcAg-staining, apolyclonal rabbit antiserum for MRP-2 detection, patient-derived serum(M. Roggendorf, Essen) for HδAg. As secondary antibodies goatanti-rabbit or -human, labelled with either AlexaFluor488 orAlexaFluor546 (Invitrogen) was used. Actin staining was performed by theaddition of atto633-labelled Phalloidin diluted 1:2000 (ATTO-Tec,Germany) to the second staining step. Images were taken on a Leica DMIRB or Leica SP2 confocal microscope (Leica, Germany), image analysiswas performed using ImageJ.

1.6 Taurocholate uptake assay: HepG2-rNtcp cells were used for studying[3H] TC uptake as described before (Kubitz et al., 2004). Briefly,HepG2-rNtcp cells were cultured for 12 h (in D-MEM/Ham's F12 w. 10% FCSmedium containing G418 for selection) were preincubated with increasingconcentrations of MyrB for 20 min before addition of TC (150 μMcontaining 450 cpm/fmol [3H]TC). Uptake was stopped after 5 min byremoving the medium and washing thrice with ice-cold PBS. Cells werelysed (0.2 M NaOH and 0.05% SDS). Radioactivity of cell lysates wasmeasured in a liquid scintillation counter (Packard instruments,Frankfurt, Germany) using Ultima Gold liquid scintillation solution(Perkin Elmer, Rodgau, Germany).

1.7 Western Blotting: Whole cell lysates were treated with PNGase F (NewEngland Biolabs) and analyzed by Western blot using rabbit anti-hNTCPantibody (Sigma-Aldrich, or the anti-serum K9.

2. Binding of Lipopetide MyrB to hNTCP

To assess whether expression of hNTCP facilitates MyrB-binding, HuH7-,HepG2-, HepaRG- and the two mouse hepatoma cells Hepa1-6 and Hep56.1Dwere stably transduced with an hNTCP-encoding lentivirus. hNTCPexpression was verified by Western Blot (FIG. 1A). HuH7^(hNTCP),HepG2^(hNTCP), Hepa1-6^(hNTCP) and Hep56.1D^(hNTCP) express comparableamounts of hNTCP. HepaRG^(hNTCP)-expression was higher for unknownreasons. No hNTCP was detected in mock-transduced cells. To examinewhether hNTCP-expression renders HuH7^(hNTCP), HepG2^(hNTCP) andHepaRG^(hNTCP) cells capable of binding HBVpreS we analysed cellassociation of atto-dye-labeled MyrB (MyrB^(atto)) by fluorescencemicroscopy (FIG. 1B) and flow cytometry (FIG. 1C). Specificity wascontrolled through MyrB-competition and the MyrB^(attoAla11-15) mutant.hNTCP-expression resulted in specific MyrB-binding indicating a validrole of hNTCP as an HBVpreS-specific receptor.

Since hepatocytes from some non-HBV susceptible species (mice(m),rats(r)) bind MyrB (Meier et al., 2012) and accumulate the peptide inthe liver after injection (Schieck et al., 2013), we expected that mNtcpand rNtcp also bind MyrB. We therefore used HepG2^(mNtcp) cells andHepG2 cells expressing a ratNTCP-eGFP-fusion and analysed MyrB^(atto)binding. We verified specific and competable binding of MyrB^(atto) toboth cell lines (FIGS. 1D and E). Taking advantage of the fluorescenceof the ratNTCP-eGFP fusion, we confirmed co-localisation of theMyrB/rNtcp-complex in microvilli.

3. Inhibition of Bile Salt Transport

3.1 Lipopeptide MyrB Inhibits the Bile Salt Transporter Function ofNTCP.

The mere size of MyrB as a specific ligand for some NTCPs suggests thatseveral contact sites are involved in binding. To test whether MyrBtherefore interferes with the bile salt transporter function of NTCPs,we analysed interference of MyrB with uptake of ³H-labeled taurocholatein Flag-rNtcp-eGFP expressing HepG2 cell lines. MyrB inhibited rNtcpwith an IC₅₀ of 4 nM (FIG. 2A). Remarkably, the IC₅₀s for inhibition ofHBV infection (˜100 pM) and of bile salt transport (˜5 nM) differsubstantially which relates to observations that infection inhibitiondoes not require binding saturation of NTCP (Schulze et al., 2010).

We furthermore analysed interference of MyrB with uptake of ³H-labeledtaurocholate in Flag-hNtcp-eGFP expressing HepG2 cell lines incomparison to two control peptides: mutant MyrB^(Ala11-15) (a mutantwith Ala mutations in the region 9-NPLGFFP-15 (amino acid positions20-26 of SEQ ID NO:2), namely 9-NPAAAAA-15 (amino acid positions 8-14 ofSEQ ID NO:21)) and preS2-78myr (see FIG. 2B).

mutant MyrB^(Ala11-15) SEQ ID NO: 21 Myr-GTNLSVPNPAAAAADHQLDPAFGANSNNPDWDFNPNKDHWPEANK VG-amide preS2-78myr SEQ ID NO: 22 Myr-ggnlstsnplgffpdhqldpafrantanpdwdfnpnkdtwpdankvgagafglgftpphggllgwsidgaggilqtlp

3.2 NTCP Substrates Taurocholate (TC), Taurodeoxycholate (TDC) andTaurochenodeoxy-Cholate (TCDC) Inhibit HBV Infection.

To test if natural substrates of NTCP affect HBV infection,differentiated HepaRG (FIG. 2C) and HuH7^(hNTCP) (FIG. 2D) cells wereinoculated with HBV at increasing concentrations of TC, TDC and TCDC.All three substrates inhibited HBV-infection at non-physiologicalconcentrations (50 μM and 500 μM) in both cell lines as shown by HBeAgsecretion. Marginal reduction was observed at 5 μM indicating that underphysiological conditions (<5 μM) hNTCP remains a functional HBV/HDVreceptor. To test if TC, TDC and TCDC interferes withMyrB^(atto)-binding we performed a binding competition assay (FIG. 2E).In the presence of 500 μM all substrates profoundly interfered withpreS-binding.

The features disclosed in the foregoing description, in the claimsand/or in the accompanying drawings may, both separately and in anycombination thereof, be material for realizing the invention in diverseforms thereof.

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The invention claimed is:
 1. A method for the treatment of acardiovascular disease (CVD) in a patient comprising administering atherapeutically effective amount of a lipopeptide to the patient,wherein the lipopeptide comprises a peptide of the general formulaX−P−Y−R ₀ wherein P is the amino acid sequence NPLGFXaaP (SEQ. ID NO:1), wherein Xaa is an arbitrary amino acid; X is an amino acid sequencehaving a length of m amino acids, wherein m is at least 4; Y is an aminosequence having a length of n amino acids, wherein n is 0 or at least 1;and wherein m+n>11; R is a C-terminal modification of said hydrophobicmodified peptide, and o is 0 or at least
 1. 2. The method of claim 1,comprising control or modification of cholesterol level or cholesteroluptake, wherein the cholesterol level or uptake is controlled ormodified by decreasing or blocking the NCTP-mediated bile salt transportby the lipopetide.
 3. The method of claim 1, wherein the route ofadministration is selected from subcutaneous, intravenous, oral, nasal,intramuscular, transdermal, inhalative, and by suppository.
 4. Themethod of claim 1, wherein the lipopeptide comprises an N-terminalhydrophobic modification.
 5. The method of claim 1, wherein thelipopetide comprises an amino acid sequence selected from SEQ ID NOs: 2to 14 and 18 to 20.